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WHO/BS/201...
WHO/BS/2013.2213
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The designations employed and the presenta...
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Table of contents

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Introduction ··························································...
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B.1.2 Product development and characterization ···············································...
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C.3.4 Manufacturing and formulation changes ··················································...
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Introduction

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These guidelines are intended to provide national regulatory authorities (NRA...
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humanized or fully human monoclonal antibodies, or antibody-related proteins or other

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engi...
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Although comprehensive characterization of the drug product is expected, considerable

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emph...
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international consultations on the development of the biosimilar guidelines and also their

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...
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Special considerations for biosimilar products are available in the WHO guidelines on

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eva...
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an indicator of a therapeutic response. A genomic biomarker is a measurable DNA and/or

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RN...
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Good laboratory practice (GLP)

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A quality system concerned with the organizational proce...
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A quantity of well-characterized cells of animal or other origin, derived from a cell seed

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Pharmacokinetics is the study and characterization of the time course of drug absorption,

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original gene is artificially altered and changed. These new gene(s) when inserted into the

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Part A. Manufacturing and quality control

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A.1 Definitions

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A.1.1 International name ...
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In this respect, rDNA-derived products are considered to be like biologicals produced by

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...
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A description of the host cell, its source and history, and of the expression vector used in
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methods used for preparation of the cell bank(s) including the cryoprotectants and media

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...
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expressing the protein from a plasmid or mammalian epigenetic expression should be

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distin...
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A.3.2.2 Cell substrate genetic stability

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The limit of in vitro cell age for production sh...
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Media and other components should comply with current WHO Guidelines on

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transmissible spo...
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Data on the consistency of culture conditions and culture growth and on the maintenance

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o...
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product for further processing should be established. Criteria for the rejection of harvests
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recommendations for the evaluation of animal cell substrates for the manufacture of

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biolo...
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cultivation method, the source materials used and the results of virus clearance studies. In
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selection of the methods used for characterization should be provided and their suitability

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clinical and nonclinical development and during stability studies should be used as the

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b...
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multi-use containers such as vials or cartridges for a pen injector, proper in-use stability
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characteristics of a rDNA-derived biotherapeutic. Consequently, the manufacturer should

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d...
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otherwise justified. For storage periods of less than 6 months, the minimum amount of

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sta...
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incorporates bracketing assumes that the stability of the intermediate condition samples is

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biotherapeutics should be undertaken. The demonstration of comparability does not

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Part B. Nonclinical evaluation

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B.1 Introduction

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The general aim of nonclinical eval...
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guidelines (30-32). Recommendations concerning timing and interplay of nonclinical and

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cl...
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impact of such changes for extrapolation of the animal findings to humans should be

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example, receptor occupancy, receptor affinity, and/or pharmacological effects, and to

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as...
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Alterations in the PK profile due to immune-mediated clearance mechanisms may affect

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the ...
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application, initially confined to the vascular system. However, with time they may

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distr...
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recovery group is included in the study, an additional minimum of two animals per sex

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wou...
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justify high dose selection, consideration should be given to the expected

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For chronic use products, repeat dose toxicity studies of 6 months duration in rodents or

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Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who ...
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Who guidelines on the quality, safety, and efficacy of biotherapeutic products prepared by recombinant dna technology who r-dna_2nd_public_consultation_28_june_2013

  1. 1. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 WHO/BS/2013.2213 ENGLISH ONLY WHO Guidelines on the Quality, Safety, and Efficacy of Biotherapeutic Products Prepared by Recombinant DNA Technology Proposed guidelines NOTE: This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on Biological Standardization (ECBS). Publication of this draft is to provide information about the proposed WHO Guidelines on the Quality, Safety, and Efficacy of Biotherapeutic Products Prepared by Recombinant DNA Technology to a broad audience and to improve transparency of the consultation process. The text in its present form does not necessarily represent an agreed formulation of the Expert Committee. Written comments proposing modifications to this text MUST be received by 20 September 2013 in the Comment Form available separately and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: Department of Essential Medicines and Health Products (EMP). Comments may also be submitted electronically to the Responsible Officer: Dr Hye-Na Kang at email: kangh@who.int. The outcome of the deliberations of the Expert Committee will be published in the WHO Technical Report Series. The final agreed formulation of the document will be edited to be in conformity with the "WHO style guide" (WHO/IMD/PUB/04.1). © World Health Organization 2013 All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whether for sale or for non-commercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-mail: permissions@who.int).
  2. 2. WHO/BS/2013.2213 Page 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors [or editors as appropriate] alone are responsible for the views expressed in this publication. Recommendations and guidelines published by WHO are intended to be scientific and advisory in nature. Each of the following sections constitutes guidance for national regulatory authorities (NRAs) and for manufacturers of biological products. If a NRA so desires, these Guidelines may be adopted as definitive national requirements, or modifications may be justified and made by the NRA. It is recommended that modifications to these Guidelines made only on condition that modifications ensure that the product is at least as safe and efficacious as that prepared in accordance with the recommendations set out below. 22 23 24
  3. 3. WHO/BS/2013.2213 Page 3 Table of contents 1 2 3 Introduction ··································································································· 6 4 Background ···································································································· 6 5 Scope ············································································································ 9 6 Glossary ······································································································ 10 7 8 Part A. Manufacturing and quality control ·························································· 16 9 A.1 Definitions ····························································································· 16 10 A.1.1 International name and proper name ·························································· 16 11 A.1.2 Descriptive definition ··········································································· 16 12 A.1.3 International standards and reference materials ············································· 16 13 A.2 General manufacturing guidelines ·································································· 16 14 A.3 Control of starting/source materials ································································ 17 15 A.3.1 Expression vector and host cell ································································ 17 16 A.3.2 Cell bank system ················································································· 18 17 A.3.3 Cell culture medium/other materials ·························································· 21 18 A.4 Control manufacturing process······································································ 22 19 A.4.1 Cell culture························································································ 22 20 A.4.2 Purification ······················································································· 24 21 A.5 Control of drug substance and drug product ····················································· 26 22 A.5.1 Characterization ·················································································· 26 23 A.5.2 Routine control ··················································································· 27 24 A.6 Filling and container ················································································· 28 25 A.7 Records, retained samples, labelling, distribution and transport ······························ 29 26 A.8 Stability, storage and expiry date ·································································· 29 27 A.8.1 Stability studies ·················································································· 29 28 A.8.2 Drug product requirements ····································································· 31 29 A.9 Manufacturing process changes ···································································· 32 30 31 Part B. Nonclinical evaluation ··········································································· 34 32 B.1 Introduction···························································································· 34 33 B.1.1 Objectives of the nonclinical evaluation ······················································ 35
  4. 4. WHO/BS/2013.2213 Page 4 1 B.1.2 Product development and characterization ··················································· 35 2 B.1.3 Good laboratory practice ········································································ 36 3 B.2 Pharmacodynamics ··················································································· 36 4 B.2.1 Primary and secondary pharmacodynamics/biological activities ························· 36 5 B.2.2 Safety pharmacology ············································································ 37 6 B.3 Pharmacokinetics/Toxicokinetics ·································································· 37 7 B.3.1 General principles ················································································ 37 8 B.3.2 Assay······························································································· 38 9 B.3.3 Distribution ······················································································· 38 10 B.3.4 Metabolism ······················································································· 39 11 B.4 Toxicity studies ······················································································· 39 12 B.4.1 General principles ················································································ 39 13 B.4.2 Single dose toxicity studies ····································································· 42 14 B.4.3 Repeat dose toxicity studies ···································································· 42 15 B.4.4 Genotoxicity studies ············································································· 43 16 B.4.5 Carcinogenicity studies ········································································· 44 17 B.4.6 Reproductive performance and developmental toxicity studies ··························· 46 18 B.4.7 Local tolerance studies ·········································································· 50 19 B.4.8 Other toxicity studies ············································································ 50 20 21 Part C. Clinical evaluation ··············································································· 54 22 C.1 Good clinical pracitice ··············································································· 54 23 C.2 Clinical pharmacology (Phase I) ··································································· 54 24 C.2.1 Initial safety and tolerability studeis ·························································· 54 25 C.2.2 Pharmacogenomics ·············································································· 56 26 C.2.3 Pharmacokinetics ················································································ 56 27 C.2.4 Pharmacodynamics ·············································································· 62 28 C.2.5 Pharmacokinetics/Pharmacodynamics relationship ········································· 62 29 C.2.6 Modifications of PK and PD profiles of therapeutic proteins ····························· 63 30 C.3 Efficacy ································································································· 63 31 C.3.1 Phase II ···························································································· 63 32 C.3.2 Confirmatory phase III ·········································································· 65 33 C.3.3 Biomarkers ······················································································· 67
  5. 5. WHO/BS/2013.2213 Page 5 1 C.3.4 Manufacturing and formulation changes ····················································· 67 2 C.3.5 Special populations ·············································································· 68 3 C.3.6 Post-marketing: Phase VI ······································································· 69 4 C.4 Statistical considerations ············································································ 69 5 C.4.1 General considerations ·········································································· 69 6 C.4.2 Special considerations for rDNA-derived biotherapeutics ································· 70 7 C.5 Safety ··································································································· 72 8 C.5.1 Special populations ·············································································· 74 9 C.6 Immunogenicity ······················································································ 75 10 C.7 Pharmacovigilance and risk managament planning ············································ 77 11 C.8 Additional guidance ·················································································· 79 12 13 Authors ········································································································ 80 14 Acknowledgements ·························································································· 84 15 References ···································································································· 85 16 17 Appendix 1. Manufacturing process validation ························································· 90 18 Appendix 2. Characterization of rDNA-derived biotherapeutics ····································· 93 19 Appendix 3. Routine control of rDNA-derived biotherapeutics ····································· 102 20 Appendix 4. Product specific guidance in nonclinical evaluation (examples) ····················· 105 21 Appendix 5. Animal species/model selection··························································· 107 22 Appendix 6. Explanatory notes ··········································································· 111 23 Appendix 7. List of abbreviations ········································································ 114 24 25 26
  6. 6. WHO/BS/2013.2213 Page 6 1 Introduction 2 These guidelines are intended to provide national regulatory authorities (NRAs) and 3 manufacturers with guidance on the quality, safety and efficacy of biotherapeutic 4 products prepared by recombinant deoxyribonucleic acid (DNA) technology (rDNA- 5 derived biotherapeutics) and intended for use in humans. They are based on experience 6 gained over the past 25 years or so in this technically demanding field and replace 7 “Guidelines for assuring the quality of pharmaceutical and biological products prepared 8 by recombinant DNA technology” (1). 9 10 Part A sets out updated guidelines for the manufacture and quality control of rDNA- 11 derived biotherapeutics, including consideration of the effects of manufacturing changes 12 and of devices used in delivery on the product and its stability. Part B is new and 13 provides guidelines on nonclinical evaluation: Part C, also new, provides guidance on 14 clinical evaluation. The nature and extent of characterization and testing (Part A) 15 required for a product undergoing nonclinical and clinical studies will vary depending on 16 the nature of the product and its stage of development . Part A may also apply to vaccines 17 prepared by rDNA technology. However, neither Part B nor C applies to the vaccines. 18 Detail guidance on nonclinical and clinical evaluation of vaccines (2, 3) as well as other 19 product specific WHO recommendations and guidelines related to vaccines are available 20 elsewhere (http://www.who.int/biologicals/vaccines/en/). 21 22 Background 23 Developments in molecular genetics and nucleic acid chemistry have enabled genes 24 encoding natural biologically active proteins to be identified, modified and transferred 25 from one organism to another so as to obtain highly efficient synthesis of their products. 26 This has led to the production of new rDNA-derived biological medicines using a range 27 of different expression systems such as bacteria, yeast, transformed cell lines of 28 mammalian origin, insect and plant cells, as well as transgenic animals. rDNA technology 29 is also used to produce non-native biologically active proteins such as chimeric,
  7. 7. WHO/BS/2013.2213 Page 7 1 humanized or fully human monoclonal antibodies, or antibody-related proteins or other 2 engineered biological medicines such as fusion proteins. 3 4 There has also been great progress in the ability to purify biologically active 5 macromolecules. In addition, analytical technologies have improved tremendously since 6 the early days of biotechnology, allowing the detailed characterization of many biological 7 macromolecules including its protein, lipid and oligosaccharide components. 8 9 Together these technologies have enabled the production of large quantities of medicinal 10 products that are difficult to prepare from natural sources or were previously unavailable. 11 Nevertheless, it is still not possible to fully predict biological properties and clinical 12 performance of these macromolecules from physicochemical characteristics alone. In 13 addition, the production processes are biological systems which are known to be 14 inherently variable, a feature which has important consequences for the safety and 15 efficacy of the resulting product. A pre-requisite, therefore, for introducing such 16 biologicals into the clinic is to ensure consistency of quality from lot to lot and for this 17 purpose robust manufacturing processes are developed based on process understanding 18 and characterization, including appropriate in-process controls. Process understanding 19 and consistency is critical since slight changes can occasionally lead to major adverse 20 effects, such as immunogenicity, with serious safety implications associated with 21 immunogenicity. 22 23 As with many other new technologies, a new set of safety issues for consideration both 24 by industry and NRAs has been generated by these particular biotechnologies. Potential 25 safety concerns arose from the novel processes used in manufacture, from product and 26 process related impurities and from the complex structural and biological properties of 27 the products themselves. Factors that have received particular attention include the 28 possible presence of contaminating oncogenic host cell DNA in products derived from 29 transformed mammalian cells (4), and the presence of adventitious viruses (4). Since the 30 nature and production of these products are highly sophisticated, they require similar 31 sophisticated laboratory techniques to ensure their proper standardization and control.
  8. 8. WHO/BS/2013.2213 Page 8 1 Although comprehensive characterization of the drug product is expected, considerable 2 emphasis must also be given to process validation and in-process control. Adequate 3 control measures relating to the starting materials and manufacturing process are, 4 therefore, as important as analysis of the drug product. Thus data on the host cell quality, 5 purity, freedom from adventitious agents, adequate in-process testing during production, 6 and effectiveness of test methods are required for licensing. 7 8 At a very early stage in the development of rDNA-derived medicines, the European 9 Medicines Agency and the US Food and Drug Administration produced guidelines and 10 points to consider, respectively, for the development and evaluation of these new 11 products (5, 6). Such guidelines, based as they were on long experience with traditional 12 biologicals, set the scene for regulatory expectations both for clinical trials and for 13 licensing. At the global level, the WHO produced a series of guidance documents on the 14 quality, safety and efficacy of rDNA-derived products, including specific guidance for 15 products such as interferons and monoclonal antibodies (1, 7-9). These regulatory 16 concepts have been instrumental in establishing the quality, safety and efficacy of rDNA- 17 derived biotherapeutics which now play a major role in today’s medical practice. 18 19 As patents and data protection measures on biotechnology products have expired, or 20 neared expiration, considerable attention has turned to producing copies of the innovator 21 products with the view to making more affordable products which may improve global 22 access to these medicines. Since by definition it is not possible to produce identical 23 biologicals, the normal method of licensing generic medicines, which relies primarily on 24 bioequivalence data, is not appropriate for licensing such products and the term similar 25 biological product, or biosimilar product, came into existence (10, 11). The concept of 26 similar biological medicinal products was introduced first by the European Medicines 27 Agency (10) and subsequently by other national regulatory authorities (although the 28 actual term used has varied slightly from agency to agency). WHO guidelines on the 29 evaluation of similar biotherapeutic products were produced in 2010 (11), and provided a 30 set of globally acceptable principles regarding the regulatory evaluation of biosimilars, 31 although it was recognized that they will not by themselves resolve all issues. During
  9. 9. WHO/BS/2013.2213 Page 9 1 international consultations on the development of the biosimilar guidelines and also their 2 implementation, it became clear that there was a need to update WHO guidance on the 3 quality, safety and efficacy of rDNA-derived medicines and biotechnology products in 4 general (12). In 2010, the International Conference of Drug Regulatory Authorities noted 5 that WHO should supplement its guidance on the evaluation of similar biotherapeutic 6 products by providing up-to-date guidelines for the evaluation of biotherapeutic products 7 in general. 8 9 The present guidelines have been developed through international consultation and are 10 intended as a replacement of those in Annex 3, TRS No 814, 1991. They are considered 11 to be a replacement and not a revision of those guidelines because they contain new 12 sections on nonclinical and clinical evaluation of rDNA-derived biotherapeutics which 13 were lacking in the original document. In addition, a section on issues related to 14 manufacturing changes both during development and once the product is on the market 15 has also been introduced since considerable improvements to the production process and 16 to the product itself can take place during the later stages of development and post 17 licensure, especially in the immediate post licensing years. These changes can 18 unintentionally impact the clinical performance of the product and need to be handled 19 carefully from a regulatory perspective. 20 21 Scope 22 These guidelines apply, in principle, to all biologically active protein products used in the 23 treatment of human diseases and which are prepared by recombinant DNA technology. 24 They also apply to protein products used in diagnosis (e.g. for monoclonal antibody 25 products including in vivo diagnosis and ex vivo treatment, but excluding in vitro 26 diagnosis) and those intentionally modified by for example pegylation or modification or 27 rDNA sequences. They set out regulatory expectations both for clinical trials and for 28 licensing, as well as for changes in products already on the market. However, the level of 29 data submitted for a product for clinical trials will have to take into account the nature of 30 the product and its stage of development. 31
  10. 10. WHO/BS/2013.2213 Page 10 1 Special considerations for biosimilar products are available in the WHO guidelines on 2 evaluation of similar biotherapeutic products, adopted by the WHO Expert Committee 3 on Biological Standardization in 2009 (11). 4 5 Although the principles outlined in this document (e.g. in Part A) apply to vaccines made 6 by rDNA technology, there are more detailed guidelines/recommendations on vaccine 7 evaluation in terms of quality, safety, and efficacy (2, 3). For example, vaccines such as 8 yeast derived hepatitis B vaccine or malaria vaccine produced by rDNA technology (13, 9 14) are available in the WHO Technical Report Series 10 (http://www.who.int/biologicals/vaccines/en/). 11 12 The present guidelines are not intended to apply to genetically modified live organisms 13 designed to be used directly in humans, such as recombinant viral vectors (15) or live 14 attenuated vaccines, nor to gene transfer products. A WHO guideline on DNA vaccines 15 for therapeutic as well as for prophylactic use, adopted by the WHO Expert Committee 16 on Biological Standardization in 2005, are available (16). Products produced in 17 transgenic animals are also excluded. 18 19 Glossary (alphabetical order) 20 The definitions given below apply to the terms used in this document. They may have 21 different meaning in other contexts. 22 23 Acceptance criteria 24 Numerical limits, ranges, or other suitable measures for acceptance of the results of 25 analytical procedures which the drug substance or drug product or materials at other 26 stages of their manufacture should meet. 27 28 Biomarkers 29 A biomarker is defined as a laboratory measurement that reflects the activity of a disease 30 process, correlates (either directly or inversely) with disease progression, and may also be
  11. 11. WHO/BS/2013.2213 Page 11 1 an indicator of a therapeutic response. A genomic biomarker is a measurable DNA and/or 2 RNA marker that measures the expression, function or regulation of a gene. 3 4 Biotherapeutic 5 A biological medicinal product with the indication of treating human diseases. 6 7 Comparability exercise 8 The activities, including study design, conduct of studies, and evaluation of data, that are 9 designed to investigate whether the products are comparable. 10 11 Critical quality attribute 12 A physical, chemical, biological or microbiological property or characteristic that is 13 selected for its ability to help indicate the consistent quality of the product within an 14 appropriate limit, range, or distribution to ensure the desired product quality. 15 16 Drug product 17 A pharmaceutical product type in a defined container closure system that contains a drug 18 substance, generally in association with excipients. 19 20 Drug substance 21 The active pharmaceutical ingredient and associated molecules that may be subsequently 22 formulated, with excipients, to produce the drug product. It may be composed of the 23 desired product, products-related substances, and product- and process-related impurities. 24 It may also contain other component such as buffers. 25 26 Good clinical practice (GCP) 27 An international ethical and scientific quality standard for designing, conducting, 28 recording and reporting trials that involve the participation of human subjects. 29 Compliance with this standard provides public assurance that the rights, safety and well- 30 being of trial subjects are protected, consistent with the principles that have their origin in 31 the Declaration of Helsinki, and that the clinical trial data are credible.
  12. 12. WHO/BS/2013.2213 Page 12 1 2 Good laboratory practice (GLP) 3 A quality system concerned with the organizational process and conditions under which 4 nonclinical health and environmental safety studies are planned, performed, monitored, 5 recorded, archived and reported. 6 7 Good manufacturing practice (GMP) 8 That part of the pharmaceutical quality assurance process which ensures that products are 9 consistently produced and to meet to the quality standards appropriate to their intended 10 use and as required by the marketing authorization. In these guidelines, GMP refers to the 11 current GMP guidelines published by WHO. 12 13 Immunogenicity 14 The ability of a substance to trigger an immune response or reaction (e.g. development of 15 specific antibodies, T cell response, allergic or anaphylactic reaction). 16 17 Impurity 18 Any component present in the drug substance or drug product that is not the desired 19 product, a product-related substance, or excipient including buffer components. It may be 20 either process- or product-related. 21 22 in-silico modeling 23 A computer-simulated model. 24 25 in-process control 26 Checks performed during production in order to monitor and, if necessary, to adjust the 27 process to ensure that the intermediate or product conforms to its specifications. The 28 control of the environment or equipment may also be regarded as a part of in-process 29 control. 30 31 Master cell bank (MCB)
  13. 13. WHO/BS/2013.2213 Page 13 1 A quantity of well-characterized cells of animal or other origin, derived from a cell seed 2 at a specific population doubling level (PDL) or passage level, dispensed into multiple 3 containers, cryopreserved, and stored frozen under defined conditions, such as the vapour 4 or liquid phase of liquid nitrogen in aliquots of uniform composition. The master cell 5 bank is prepared from a single homogeneously mixed pool of cells. 6 7 Non-human primates (NHPs) 8 Primates used as models for the study of the effects of drugs in humans, prior to clinical 9 studies. 10 11 P450 (CYP) enzymes 12 Indicates the family of metabolising enzymes which is the most common group. 13 14 Pharmacodynamics (PD) 15 The study of the biochemical and physiological effects of drugs on the body and the 16 mechanisms of drug action and the relationship between drug concentration and effect. 17 One dominant example is drug-receptor interactions. PD is often summarized as the study 18 of what a drug does to the body, as opposed to pharmacokinetics which is the study of 19 what the body does to a drug. 20 21 Pharmacogenomics 22 The study of the pharmacologic correlation between drug response and variations in 23 genetic elements has become of increasing importance for drug development. Such 24 variations can have effects on the risk of developing adverse drug reactions as well as on 25 the response to treatment; variations in drug pharmacokinetics and metabolic pathways 26 can cause higher drug concentrations in some patients resulting in increased drug toxicity, 27 and/or lower drug concentrations in some patients resulting in decreased drug effects. 28 29 Pharmacokinetics (PK)
  14. 14. WHO/BS/2013.2213 Page 14 1 Pharmacokinetics is the study and characterization of the time course of drug absorption, 2 distribution, metabolism, and elimination (ADME). Pharmacokinetics is a quantitative 3 analysis of how living systems handle foreign compounds. 4 5 Pharmacovigilance (PhV) 6 The activities that are carried out after a medicinal product is marketed to observe and 7 manage in a continuous manner the safety and the efficacy of the products. 8 9 QT/QTc 10 QT interval is a measure of the time between the start of the Q wave and the end of the T 11 wave in the heart's electrical cycle on the electrocardiogram. It measures the conduction 12 speed between the atria and the ventricles. There is a genetic predisposition to the 13 prolongation of the QT interval which can be triggered by several factors, including 14 various medicinal products by themselves or due to their metabolic interaction. It is 15 critical to understand whether a particular drug or a biological trigger the prolongation, as 16 any prolongation of the QT interval outside of the normal limits determined for 17 electrocardiograms indicates potential for arrhythmia (disturbed heart rhythm) which is a 18 serious adverse event during drug therapy. In extreme cases, this can lead to sudden death. 19 Since the QT interval is affected by the heart rate, “corrected” QT-QTc should also be 20 used. 21 22 rDNA-derived biotherapeutics 23 Biotherapeutics prepared by recombinant DNA technology. All biologically active 24 protein products used in the treatment of human diseases and which are prepared by 25 rDNA technology. These include recombinant protein biotherapeutics, recombinant blood 26 products, recombinant monoclonal antibodies and recombinant enzymes. 27 28 Recombinant DNA technology 29 Technology joining together (recombine) DNA segments from two or more different 30 DNA molecules that are inserted into a host organism to produce new genetic 31 combinations. Also referred to as gene manipulation or genetic engineering, as the
  15. 15. WHO/BS/2013.2213 Page 15 1 original gene is artificially altered and changed. These new gene(s) when inserted into the 2 expression system form the basis for the production of rDNA-derived protein(s). 3 4 Risk management plan (RMP) 5 The activities that will, in a continuous manner ensure that patients continue to be safe 6 and benefit from a medicinal ingredient. These plans include PhV plans amongst many 7 other elements. 8 9 Source material/starting material 10 Any substance of a defined quality used in the production of a biological medicinal 11 product, but excluding packaging materials. 12 13 Specification 14 A list of tests, references to analytical procedures, and appropriate acceptance criteria 15 which are numerical limits, ranges, or other criteria for the tests described. Specifications 16 are critical quality standards that are proposed and justified by the manufacturer and 17 approved by regulatory authorities. 18 19 Working cell bank (WCB) 20 A quantity of well-characterized cells of animal or other origin, derived from the master 21 cell bank at a specific PDL or passage level, dispensed into multiple containers, 22 cryopreserved, and stored frozen under defined conditions, such as in the vapour or liquid 23 phase of liquid nitrogen in aliquots of uniform composition. The working cell bank is 24 prepared from a single homogeneously mixed pool of cells. One or more of the WCB 25 containers is used for each production culture. 26
  16. 16. WHO/BS/2013.2213 Page 16 1 2 Part A. Manufacturing and quality control 3 A.1 Definitions 4 A.1.1 International name and proper name 5 Where an International Non-Proprietary Name (INN) for a rDNA-derived biotherapeutic 6 is available, it should be used (17). The proper name should be the equivalent of the INN 7 in the language of the country of origin. 8 9 A.1.2 Descriptive definition 10 The description of a rDNA-derived biotherapeutic should indicate the biological system 11 in which it is produced (e.g. bacterial, fungal or mammalian cells) as well as the 12 presentation of the drug product. 13 14 A.1.3 International standards and reference materials 15 International standards and reference preparations have been established for a wide range 16 of biologicals prepared by rDNA technology. They are used to calibrate assays either 17 directly or for calibration of secondary standards or manufacturers working standards. A 18 list of such materials is available on WHO website 19 (http://www.who.int/bloodproducts/catalogue/AlphFeb2013.pdf). Each 20 standard/reference preparation is held by one of the WHO custodian laboratories, e.g. the 21 National Institute for Biological Standards and Control, Potters Bar, United Kingdom. 22 23 A.2 General manufacturing guidelines 24 The present Guidelines cover the following three main areas: 25 26 1) Control of starting/source materials, including data both on the host cell and on the source, nature and sequence of the gene used in production. 27 2) Control of the manufacturing process. 28 3) Control of the drug substance and the drug product. 29
  17. 17. WHO/BS/2013.2213 Page 17 1 In this respect, rDNA-derived products are considered to be like biologicals produced by 2 traditional methods, such as bacterial and viral vaccines, where the quality, safety and 3 efficacy of the product relies heavily on adequate control of the starting/source materials 4 and on the manufacturing process, in addition to control tests on the drug substance and 5 drug products themselves. These guidelines therefore place considerable emphasis on the 6 characterization and testing of host cell lines and other materials used during 7 manufacturing and in validating the ability of the purification processes to remove or 8 inactivate unwanted materials, especially possible viral contaminants and process related 9 impurities such as proteins and DNA. They also cover in-process controls in 10 manufacturing and comprehensive characterization of the drug substance and the drug 11 product. 12 13 Information should therefore be provided to adequately describe the starting/source 14 materials, manufacturing process and in-process controls. The description of the 15 manufacturing process should be provided in the form of a flow diagram and sequential 16 procedural narrative and the in-process controls for each step or stage of the process 17 should be indicated in this description. Also, an explanation should be provided of how 18 batches of the drug substance and drug product are defined (e.g. splitting and pooling of 19 harvests or intermediates). Details of batch size or scale and batch numbering should also 20 be included. 21 22 The general recommendations for manufacturing establishments contained in the WHO 23 Good manufacturing practices: main principles for pharmaceuticals preparations (18) 24 and the Good manufacturing practices for biological products (19) as well as those in the 25 WHO Recommendations for the evaluation of animal cell cultures as substrates for the 26 manufacture of biological medicinal products and for the characterization of cell banks 27 (4) should apply to establishments manufacturing rDNA-derived biotherapeutics. 28 29 A.3 Control of starting/source materials 30 A 3.1 Expression vector and host cell
  18. 18. WHO/BS/2013.2213 Page 18 1 A description of the host cell, its source and history, and of the expression vector used in 2 production, including source and history, should be given. This should include details of 3 the origin and identity of the gene being cloned as well as the construction, genetic 4 elements contained and structure of the expression vector. An explanation of the source 5 and function of the component parts of the vector, such as the origins of replication, 6 promoters, or antibiotic markers, should be provided as well as a restriction-enzyme map 7 indicating at least those sites used in construction. 8 9 Methods used to amplify the expression constructs, transform expression constructs into 10 host cells, and rationale used to select the cell clone for production should be fully 11 described. The vector within the cell, whether integrated or extrachromosomal, and copy 12 number, should be analyzed. A host cell containing an expression vector should be cloned 13 and used to establish a master cell bank (MCB) and the correct identity of the vector 14 construct in the cell bank should be established. The genetic stability of the host-vector 15 combination should be documented (see below). 16 17 The nucleotide sequence of the cloned gene insert and of the flanking control regions of 18 the expression vector should be indicated and all relevant expressed sequences clearly 19 delineated. 20 21 Measures to promote and control the expression of the cloned gene in the host cell during 22 production should be described in detail. 23 24 A.3.2 Cell bank system 25 Typically, rDNA-derived biotherapeutics are produced using a cell bank system which 26 involves a manufacturer’s working cell bank (WCB) derived from a MCB. It is 27 acknowledged that a WCB may not always be established in early phases of 28 development. 29 30 The type of banking system used, the size of the cell bank(s), it’s life expectancy, the 31 container (vials, ampoules, or other appropriate vessels) and closure system used, the
  19. 19. WHO/BS/2013.2213 Page 19 1 methods used for preparation of the cell bank(s) including the cryoprotectants and media 2 used, and the conditions employed for cryopreservation or long term storage conditions 3 should all be documented and described in detail. 4 5 Evidence for banked cell stability under defined storage conditions should be provided. 6 Such evidence can be generated during production of material from the banked cells and 7 supported by a programme for monitoring stability indicating attributes over time (e.g. 8 data on cell viability upon thawing, stability of the host-vector expression system in the 9 cell bank). Available data should be clearly documented and the proposed stability 10 monitoring programme described in the marketing application. Evidence for the stability 11 of the host-vector expression in the cell bank under storage as well as under recovery 12 conditions should be provided. 13 14 For animal cells and animal derived cell banks, reference should be made to the WHO 15 Recommendations for the evaluation of animal cell cultures as substrates for the 16 manufacture of biological medicinal products and for the characterization of cell banks 17 (4). 18 19 A.3.2.1 Control of cell banks 20 The characterization and testing of banked cell substrates is a critical component of the 21 control of rDNA-derived biotherapeutics. Cell banks should be tested to confirm the 22 identity, purity, and suitability of the cell substrate for the intended manufacturing use. 23 The MCB should be characterized for relevant phenotypic and genotypic markers which 24 should include the expression of the recombinant protein and/or presence of the 25 expression construct. The testing program chosen for a given cell substrate will vary 26 according to the nature and biological properties of the cells (e.g. growth requirements) 27 and its cultivation history (including use of human-derived or animal-derived biological 28 reagents). The extent of characterization of a cell substrate may influence the type or 29 level of routine testing needed at later stages of manufacturing. Molecular methods 30 should be used to analyse the expression construct for copy number, for insertions or 31 deletions, and for the number of integration sites. Requirements for bacterial systems
  20. 20. WHO/BS/2013.2213 Page 20 1 expressing the protein from a plasmid or mammalian epigenetic expression should be 2 distinguished from mammalian cell systems. The nucleic acid sequence should be shown 3 to be identical to that determined for the expression construct and should correspond to 4 that expected for the protein sequence. 5 6 Animal cell substrates are subject to contamination and have the capacity to propagate 7 extraneous, adventitious organisms, such as mycoplasma and viruses. In addition, animal 8 cells contain endogenous agents such as retroviruses that may raise safety concerns. 9 Testing of cell substrates for both endogenous (e.g. retroviruses) and adventitious agents 10 is critical. A strategy for testing cell banks for adventitious agents should be developed. 11 This strategy should also involve an assessment of specific viruses and the families of 12 viruses that may potentially contaminated the cell substrate. Such testing is described in 13 detail in the WHO Recommendations for the evaluation of animal cell cultures as 14 substrates for the manufacture of biological medicinal products and for the 15 characterization of cell banks (4) and the International Conference on Harmonization 16 (ICH) guidelines Q5A viral safety evaluation of biotechnology products derived from cell 17 lines of human or animal origin (20). 18 19 In general, cell substrates contaminated with microbial agents are not suitable for 20 production. However, there are exceptions. For example, some murine cell lines that are 21 widely used for the production of rDNA-derived biotherapeutics express endogenous 22 retroviral particles. In such circumstances, risk mitigating strategies should be 23 implemented. These include removal of such agents and/or their inactivation by physical, 24 enzymatic and/or chemical treatment during processing of the rDNA-derived 25 biotherapeutics. 26 27 In addition, tests of purity and limited tests of identity should be performed once on each 28 WCB. For the WCB, a specification including test methods and acceptance criteria 29 should be established. A protocol for establishing future WCB should be provided. Each 30 new WCB should comply with the established WCB specification. 31
  21. 21. WHO/BS/2013.2213 Page 21 1 A.3.2.2 Cell substrate genetic stability 2 The limit of in vitro cell age for production should be defined by the time of registration, 3 and based on data derived from production cells expanded under pilot plant scale or 4 commercial scale conditions to the proposed limit of in vitro cell age for production use 5 or beyond. Generally, the production cells are obtained by expansion of cells from the 6 WCB. 7 8 Specific traits of cells, which may include, for example, morphological characteristics, 9 growth characteristics, biochemical markers, immunological markers, productivity of the 10 desired product, or other relevant genotypic or phenotypic markers may be useful for the 11 assessment of cell substrate stability during culture phase. The nucleotide sequence of the 12 insert encoding the rDNA-derived biotherapeutic should be determined at least once after 13 a full-scale culture for each MCB. 14 15 In some cases, multiple harvests from long fermentations could lead to a drift in some 16 quality attributes such as glycosylation, with the appearance of "new" variants with 17 possible impact on quality, safety and efficacy of the product. The management of such 18 drift should be appropriately addressed in process evaluation/validation studies. The 19 molecular integrity of the gene being expressed and the phenotypic and genotypic 20 characteristics of the host cell after long-term cultivation should be established and 21 defined by the time of registration. 22 23 A.3.3 Cell culture medium/other materials 24 Materials used in the manufacture of the drug substance (e.g. solvents, reagents, 25 enzymes) should be listed identifying where each material is used in the process. 26 Information on the source, quality and control of these materials should be provided. 27 Information demonstrating that materials (including biologically-sourced materials, e.g. 28 media components, monoclonal antibodies, enzymes) meet standards appropriate for their 29 intended use (including the clearance or control of adventitious agents) and for the cases 30 when suppliers of materials change should be provided, as appropriate. 31
  22. 22. WHO/BS/2013.2213 Page 22 1 Media and other components should comply with current WHO Guidelines on 2 transmissible spongiform encephalopathies in relation to biological and pharmaceutical 3 products (21). The latest version of the WHO Guidelines on tissue infectivity distribution 4 in transmissible spongiform encephalopathies (22) should also be consulted. These tables 5 are periodically updated as new data becomes available (e.g. 23). 6 7 A.4 Control of the manufacturing process 8 Adequate design of a process and knowledge of its capability are part of the strategy used 9 to develop a manufacturing process which is controlled and reproducible, yielding a drug 10 substance and drug product that consistently meet specifications. In this respect, limits 11 are justified based on information gained from the entire process from early development 12 through commercial scale production. 13 In-process controls are performed at critical decision making steps and at other steps 14 where data serve to ensure the appropriate performance of the manufacturing process, and 15 to demonstrate adequate quality during the production of both the drug substance and the 16 drug product. Those process parameters that are found to impact the quality attributes of 17 the drug substance or drug product should be controlled by suitable acceptance limits. 18 Where appropriate, in-process controls may alleviate the need for routine testing of some 19 quality attribute(s) at the level of the drug substance and/or drug product. 20 21 A.4.1 Cell culture 22 A 4.1.1 Production at finite passage 23 Procedures and materials used both for cell growth and for the induction of the product 24 should be described in detail. For each production run, data on the extent and nature of 25 any microbial contamination of culture vessels should be provided. Acceptable limits for 26 potential contamination should be set and the sensitivity of the methods used to detect it 27 indicated. Microbial and fungal contamination should be monitored according to Part A 28 Section 5.2 of General requirements for the sterility of biological substances (24) or by 29 methods approved by the NRA. 30
  23. 23. WHO/BS/2013.2213 Page 23 1 Data on the consistency of culture conditions and culture growth and on the maintenance 2 of product yield should be presented. Criteria for the rejection of culture lots should be 3 established. The maximum number of cell doublings or passage levels to be permitted 4 during production should be specified taking into account the limit of in-vitro cell age. 5 For a process demonstrating consistent growth characteristics over the proposed cell age 6 range for production, it may also be acceptable to define the cell age limit based on the 7 maximum permitted days in culture from thaw to end of production. 8 9 Host-cell / vector characteristics at the end of production cycles should be monitored to 10 establish consistency, for which purpose information on plasmid copy number or degree 11 of retention of the expression vector within the host cell may be of value, as may 12 restriction enzyme mapping of the vector containing the gene insert. If the vector is 13 present in multiple copies integrated into the host cell genome, confirming the rDNA 14 sequence directly may be difficult. In such cases, alternative approaches to confirming 15 the sequence of insert encoding the rDNA-derived biotherapeutics should be considered 16 and defined by the time of registration (e.g. restriction fragment length polymorphism 17 (RFLP), fluorescence in situ hybridization (FISH), polymerase chain reaction-single- 18 strand conformation polymorphism (PCR-SSCP), Southern Blot). For example, 19 confirmation of protein sequence by peptide mapping is an appropriate alternative to 20 rDNA sequencing. 21 22 A.4.1.2 Continuous culture production 23 As recommended above, all procedures and materials used for cell culture and induction 24 of the product should be described in detail. In addition, particular consideration should 25 be given to the procedures used in production control. Monitoring is necessary 26 throughout the life of the culture, although the frequency and type of monitoring required 27 depend on the nature of both the production system and product. 28 29 Evidence should be produced to show that variations in yield or other culture parameters 30 do not exceed specified limits. The acceptance of harvests for further processing should 31 be clearly linked to the monitoring schedule in use, and a clear definition of “batch” of
  24. 24. WHO/BS/2013.2213 Page 24 1 product for further processing should be established. Criteria for the rejection of harvests 2 or termination of the culture should also be established. Tests for microbial contamination 3 should be performed as appropriate to the harvesting strategy. 4 5 The maximum period of continuous culture should be specified, based on information on 6 the stability of the system and consistency of the product during and after this period. In 7 long-term continuous culture, the cell line and product should be fully re-evaluated at 8 intervals determined by information on the stability of the host-vector system and the 9 characteristics of the product. 10 11 A.4.2 Purification 12 The methods used for harvesting, extraction and purification of the product and related 13 in-process controls, including their acceptance criteria, should be described in detail. 14 Special attention should be given to the elimination of viruses, nucleic acid, host cell 15 proteins and impurities considered to pose an immunogenicity risk. 16 17 The ability of the purification procedure to remove unwanted product-related or process- 18 related impurities (e.g. host-cell derived proteins, nucleic acid, carbohydrates, viruses and 19 other impurities, including media derived compounds and undesirable chemicals 20 introduced by the purification process itself) should be investigated thoroughly, as should 21 the reproducibility of the process. Particular attention should be given to demonstrating 22 the removal and/or inactivation of possible contaminating viruses and residual DNA from 23 products manufactured using continuous cell lines. 24 25 A.4.2.1 Residual cellular DNA from continuous cell lines (rcDNA) 26 The ability of the manufacturing process to reduce the amount of rcDNA to an acceptable 27 level, to reduce the size of the rcDNA or to chemically inactivate the biological activity 28 of this DNA should be demonstrated. 29 30 Acceptable limits on the amount of rcDNA as well as points to be considered concerning 31 the size of rcDNA in a rDNA- derived biotherapeutic are discussed in WHO
  25. 25. WHO/BS/2013.2213 Page 25 1 recommendations for the evaluation of animal cell substrates for the manufacture of 2 biological medicinal products and for the characterization of cell banks (4). These should 3 be set taking into consideration the characteristics of the cell substrate, the intended use 4 and route of administration of the rDNA-derived biotherapeutics and, most importantly, 5 the effect of the manufacturing process on the size, quantity and biological activity of the 6 residual host cell DNA fragments. In general it has been possible to reduce rcDNA levels 7 in rDNA-derived biotherapeutics to <10 ng per dose. 8 9 A.4.2.2 Virus clearance 10 For cell substrates of human or animal origin, virus clearance or inactivation processes, 11 individually and overall, should be shown to be able to adequately remove/inactivate any 12 contaminating viruses and to ensure viral safety in the drug substance. 13 14 Where appropriate, validation studies (see Appendix 1) should be undertaken using small 15 scale studies with carefully selected model viruses to evaluate the virus 16 clearance/inactivation capability of selected process steps and overall, aiming at a 17 significant safety margins. The results will indicate the extent to which these 18 contaminants can theoretically be inactivated and removed during purification. 19 20 The overall manufacturing process, including the testing and selection of the cells and 21 source materials, as well as the validation of the ability of the purification process to 22 adequately remove possible contaminants, should ensure the absence of infectious agents 23 in the drug product. Nevertheless, to complement such approaches, testing of the product 24 itself at appropriate steps in the production process for the absence of contaminating 25 infectious viruses is also recommended. A sample of the unprocessed bulk following 26 fermentation constitutes one of the most suitable levels at which the possibility of 27 detecting adventitious virus contamination can be determined with a high probability of 28 detection. A programme of ongoing assessment of adventitious viruses in production 29 batches should be undertaken. The scope, extent and frequency of virus testing on the 30 unprocessed bulk should take into account the nature of the cell lines used, the results and 31 extent of virus testing performed during the qualification of the MCB and WCB, the
  26. 26. WHO/BS/2013.2213 Page 26 1 cultivation method, the source materials used and the results of virus clearance studies. In 2 vitro screening tests using one or more cell lines are generally used to test unprocessed 3 bulk. If appropriate, a PCR test or other suitable methods may be used. 4 5 If contamination by adventitious viruses is detected in the unprocessed bulk, the 6 manufacturing process should be carefully checked to determine the cause of the 7 contamination and to decide on the appropriate action to take. 8 9 Further considerations of the detection, elimination and inactivation of viruses in animal 10 cell substrates used in the production of rDNA-derived biotherapeutics, as well as the 11 problem of rcDNA, can be found in the WHO Recommendations for the evaluation of 12 animal cell cultures as substrates for the manufacture of biological medicinal products 13 and for the characterization of cell banks (4) as well as in the ICH guidelines Q5A viral 14 safety evaluation of biotechnology products derived from cell lines of human or animal 15 origin (20). 16 17 A.5 Control of drug substance and drug product 18 A.5.1 Characterization 19 Rigorous characterization of the rDNA-derived biotherapeutics by chemical, 20 physicochemical and biological methods is essential. Characterization is typically 21 performed in the development phase to determine the physicochemical properties, 22 biological activity, immunochemical properties, purity and impurities of the product, and 23 following significant process changes and/or for periodic monitoring to confirm the 24 quality of the product. Characterization allows relevant specifications to be established. 25 26 Particular attention should be given to using a wide range of analytical techniques 27 exploiting different physiochemical properties of the molecule (size, charge, isoelectric 28 point, amino acid composition, hydrophobicity). Post-translational modifications, such as 29 glycosylation should be identified and adequately characterized. It may also be necessary 30 to include suitable tests to establish that the product has the desired conformation, state of 31 aggregation and/or degradation, as well as higher order structure. The rationale for
  27. 27. WHO/BS/2013.2213 Page 27 1 selection of the methods used for characterization should be provided and their suitability 2 should be justified bearing in mind that the characterization of the product is intended to 3 identify attributes that may be important to the overall safety and efficacy of the product. 4 Details of the expected characterization of a rDNA-derived biotherapeutic and techniques 5 suitable for such purposes are set out in Appendix 2. The specific technical approach 6 employed will vary from product to product and alternative approaches, other than those 7 included in the appendix, will be appropriate in many cases. New analytical technologies 8 and modifications to existing technologies are continuously being developed and should 9 be utilized when appropriate. 10 11 Where relevant and possible, characteristics of the properties of the product should be 12 compared with its natural counterpart. For example, post-translational modifications, 13 such as glycosylation are likely to differ from those found in the natural counterpart and 14 may influence the biological, pharmacological and immunological properties of the 15 rDNA-derived biotherapeutics. 16 17 A.5.2 Routine control 18 Not all the characterization and testing described above in A 5.1 and in Appendix 2, 19 needs to be carried out on each batch of drug substance and drug product prior release on 20 the market. Some tests may need to be performed only initially and/or periodically to 21 establish or verify the validity or acceptability of a product and its manufacturing process. 22 Others may be required on a routine basis. A comprehensive analysis of the initial 23 production batches is expected in order to establish consistency with regard to identity, 24 purity and potency. A more limited series of tests is appropriate for routine control as 25 outlined below and in more detail in Appendix 3. Tests for use in routine control should 26 be chosen to confirm quality. The rationale and justification for including and/or 27 excluding testing for specific quality attributes should be provided. 28 29 An acceptable number of consecutive batches should be characterized to determine 30 consistency of analytical parameters. Any differences between one batch and another 31 should be noted. Data obtained from such studies as well as knowledge gained from
  28. 28. WHO/BS/2013.2213 Page 28 1 clinical and nonclinical development and during stability studies should be used as the 2 basis for establishing product specifications. 3 4 The selection of tests to be included in the routine control programme will be product 5 specific and should take into account the quality attributes (e.g. potential influence on 6 safety, efficacy or stability), the process performance (e.g. clearance capability, content), 7 the controls in place through the manufacturing process (e.g. multiple testing points), and 8 the material used in relevant nonclinical and clinical studies. These tests should include 9 criteria such as potency, the nature and quantity of product-related substances, product- 10 related impurities, process-related impurities, and absence of contaminants. 11 12 A.6 Filling and container 13 The general requirements concerning filling and containers given in the WHO Guidelines 14 on good manufacturing practices for biological products (19) should apply. 15 16 A description of the container closure systems for the drug substance and the drug 17 product should be provided including a specification for their component materials. 18 Evidence exists to show that formulated proteins can interact chemically with the 19 formulation excipients and/or the container closure system, and can, for example, lead to 20 the formation of potentially immunogenic complexes. The suitability of the container 21 closure system should be evaluated and described for its intended use. This should cover 22 evaluation of the compatibility of the materials of construction with the formulated 23 product, including adsorption to the container, leaching and other chemical or physical 24 interaction between the product and the contacting materials. The integrity of the closure 25 and its ability to protect the formulation from contamination and maintain sterility needs 26 to be ensured. 27 28 When a delivery device is presented as part of the drug product (e.g. prefilled syringe, 29 single use autoinjector), it is important to demonstrate the functionality of such a 30 combination, such as the reproducibility and accuracy of the dispensed dose under testing 31 conditions which should simulate the use of the drug product as closely as possible. For
  29. 29. WHO/BS/2013.2213 Page 29 1 multi-use containers such as vials or cartridges for a pen injector, proper in-use stability 2 studies should be performed to evaluate the impact of the in-use period of the vial or the 3 assembled device on the formulation and the functionality of the pen injector. Dose 4 accuracy should be demonstrated for the first and last dose delivered. In addition, the 5 effect of multiple injections/withdrawals on the closure should be evaluated. 6 7 A.7 Records, retained samples, labelling, distribution and transport 8 The requirements given in the WHO Guidelines on good manufacturing practices for 9 biological products (19) should apply. 10 11 The conditions of shipping should be such as to ensure that the products are maintained at 12 the appropriate environment. 13 14 A.8 Stability, storage and expiry date 15 A.8.1 Stability studies 16 For proteins, maintenance of biological activity is generally dependent on maintaining 17 molecular conformation. Such products can be particularly sensitive to environmental 18 factors such as temperature changes, oxidation, and light exposure. In order to ensure 19 maintenance of biological activity and to avoid degradation, appropriate conditions for 20 their storage are usually necessary. 21 22 A detailed protocol for the assessment of the stability of both drug substance and drug 23 product in support of the proposed storage conditions and expiration dating periods 24 should be developed. This should include all necessary information which demonstrates 25 the stability of the rDNA-derived biotherapeutics throughout the proposed shelf life 26 including, for example, well-defined specifications and test intervals. 27 28 Each product should retain its specification within established limits for stability- 29 indicating attributes, including potency throughout its proposed shelf-life. Specifications 30 should be derived from all available information using appropriate statistical methods. 31 There is no single stability-indicating assay or parameter that profiles the stability
  30. 30. WHO/BS/2013.2213 Page 30 1 characteristics of a rDNA-derived biotherapeutic. Consequently, the manufacturer should 2 develop a stability-indicating programme that provides assurance that changes in the 3 quality and potency of the product will be detected. 4 5 Primary data to support a requested storage period for either drug substance or drug 6 product should be based on long-term, real-time, and real-condition stability studies, 7 covering up to or beyond the claimed shelf-life. In cases where the stability of the product 8 is influenced by storage of intermediates (e.g. significant degradation trend observed 9 during storage of an intermediate), cumulative stability study should be considered. This 10 study should include all intermediates stored at the longest storage time claimed, or 11 selection of the most storage sensitive intermediates, as appropriate. Considering the time 12 necessary to generate the data, such cumulative study could be presented and justified in 13 the proposed stability programme at the time of licensing. 14 Also, stability studies should include an evaluation of the impact of the container closure 15 system on the formulated rDNA- derived biotherapeutics throughout the shelf life. In 16 order to ensure that the formulated product is in contact with all material of the container 17 closure system, stability studies should include samples maintained in the inverted or 18 horizontal position (i.e. in contract with the closure), as well as in the upright position, to 19 determine the effects of the closure on product quality. Data should be supplied for all 20 different container closure combinations that will be marketed. 21 22 Stability information should be provided on at least 3 batches for which manufacture and 23 storage are representative of the commercial process. 24 25 When shelf-lives of 1 year or less are proposed, real-time stability studies should be 26 conducted monthly for the first 3 months and at 3 month intervals thereafter. For products 27 with proposed shelf-lives of greater than 1 year, the studies should be conducted every 3 28 months during the first year of storage, every 6 months during the second year, and 29 annually thereafter. A minimum of 6 months data at the time of submission should be 30 submitted in cases where storage periods greater than 6 months are requested, unless
  31. 31. WHO/BS/2013.2213 Page 31 1 otherwise justified. For storage periods of less than 6 months, the minimum amount of 2 stability data in the initial submission should be determined on a case-by-case basis. 3 4 It is recommended that stability studies under accelerated and stress conditions, including 5 the impact of the container closure system (see A.6), should also be conducted on the 6 drug product. Studies under accelerated conditions may provide useful supportive data 7 for establishing the expiry date, provide product stability information for future product 8 development (e.g. preliminary assessment of proposed manufacturing changes such as 9 changes in formulation or scale-up), assist in validation of analytical methods for the 10 stability program, or generate information which may help elucidate the degradation 11 profile of the rDNA-derived biotherapeutics. Studies under stress conditions may also be 12 useful in determining whether accidental exposures to conditions other than those 13 proposed (e.g. during transportation) are deleterious to the product and for evaluating 14 which specific test parameters may be the best indicators of product stability. 15 16 Further guidance on both general and specific aspects of stability testing of a rDNA - 17 derived biotherapeutic can be obtained by consulting the WHO guidelines on the stability 18 testing of active pharmaceutical ingredients and finished pharmaceutical products (25), 19 as well as the WHO Guidelines for stability evaluation of vaccines (26). 20 21 A.8.2 Drug product requirements 22 Stability information should be provided on at least 3 batches of drug product 23 representative of that which will be used in commercial manufacture, and presented in the 24 final container. Where possible, the drug product batches included in stability testing 25 should be derived from different batches of drug substance. 26 27 Where one product is distributed in multiple presentations, the samples to be entered into 28 the stability program may be selected on the basis of a matrix system and/or by 29 bracketing. Where the same strength and exact container/closure system is used for 3 or 30 more fill contents, the manufacturer may elect to place only the smallest and largest 31 container size into the stability program, i.e. bracketing. The design of a protocol that
  32. 32. WHO/BS/2013.2213 Page 32 1 incorporates bracketing assumes that the stability of the intermediate condition samples is 2 represented by those at the extremes. In certain cases, data may be needed to demonstrate 3 that all samples are properly represented by data collected for the extremes. 4 Matrixing, i.e. the statistical design of a stability study in which account is taken of 5 factors such as the tests, process characteristics, presentation characteristics and different 6 testing time points, should only be applied when appropriate documentation is provided 7 that confirms that the stability of the samples tested represents the stability of all samples. 8 The differences in the samples for the same drug product should be identified as, for 9 example, covering different batches, different strengths, different sizes of the same 10 closure and possibly, in some cases, different container/closure systems. Matrixing 11 should not be applied to samples with differences that may affect stability, such as 12 different strengths and different containers/closures, where it cannot be confirmed that 13 the products respond similarly under storage conditions. 14 15 For preparations intended for use after reconstitution, dilution or mixing, in-use stability 16 data should be obtained. The stability should be demonstrated up to and beyond the 17 storage conditions and the maximum storage period claimed. 18 19 In addition to the standard data necessary for a conventional single-use vial, it should be 20 shown that the closure used with a multiple-dose vial is capable of withstanding the 21 conditions of repeated insertions and withdrawals so that the product retains its identity, 22 strength, potency, purity, and quality for the maximum period specified in the 23 instructions-for-use on containers, packages, and/or package inserts. 24 25 A.9 Manufacturing process changes 26 Changes to the manufacturing processes of a rDNA-derived biotherapeutic often occur 27 both during development and after approval. The reasons for such changes include 28 improving the manufacturing process, increasing scale, site change, improving product 29 stability, or complying with changes in regulatory requirements. When substantial 30 changes are made to the manufacturing process, a comparability exercise to evaluate the 31 impact of the change(s) on the quality, safety and efficacy of the rDNA-derived
  33. 33. WHO/BS/2013.2213 Page 33 1 biotherapeutics should be undertaken. The demonstration of comparability does not 2 necessarily mean that the quality attributes of the pre-change and post-change product are 3 identical, but that they are highly similar and that the existing knowledge is sufficiently 4 predictive to ensure that any differences in quality attributes have no adverse impact upon 5 safety or efficacy of the rDNA-derived biotherapeutics. The reason for each significant 6 change should be explained, together with an assessment of its potential to impact on 7 quality, safety and efficacy. 8 9 The extent of a comparability exercise depends on the potential impact of the process 10 change(s) on the quality, safety and efficacy of the product. It can range from analytical 11 testing alone (e.g. where process changes lead to no changes in any quality attribute) to a 12 comprehensive exercise requiring nonclinical and clinical bridging studies (e.g. the 13 establishment of a new host cell line with altered properties resulting in more pronounced 14 changes in quality attributes). If assurance of comparability can be shown through 15 analytical studies alone, nonclinical or clinical studies with the post-change product may 16 not be necessary. However, where the relationship between specific quality attributes and 17 safety and efficacy has not been established, and differences between quality attributes of 18 the pre- and post-change product are observed, it might be appropriate to include a 19 combination of quality, nonclinical, and/or clinical studies in the comparability exercise. 20 21 Further considerations of manufacturing changes can be found in guidelines provided by 22 the ICH (27), the European Medicines Agency (EMA) (28), the United States Food and 23 Drug Administration (US FDA) (29) and other major NRAs. 24
  34. 34. WHO/BS/2013.2213 Page 34 1 Part B. Nonclinical evaluation 2 3 B.1 Introduction 4 The general aim of nonclinical evaluation is to determine whether new medicinal 5 products possess the desired pharmacodynamic (PD) activity and have the potential to 6 cause unexpected and undesirable effects. However, classical PD, safety or toxicological 7 testing, as recommended for chemical drugs, may be of only limited relevance for rDNA- 8 derived biotherapeutics due to their unique and diverse structural and biological 9 properties including species specificity, immunogenicity, and unpredicted pleiotropic 10 activities. These properties pose particular problems in relation to nonclinical testing in 11 animals, and their pharmacological and safety evaluation will have to take a large number 12 of factors into account. Thus, a flexible approach is necessary for the nonclinical 13 evaluation of rDNA-derived biotherapeutics. For example, certain proteins, e.g. 14 interferons, are highly species-specific, so that the human protein is pharmacologically 15 much more active in humans than in any animal species. Furthermore, human proteins 16 frequently produce immunological responses in animal species which may ultimately 17 modify their biological effects and may result in toxicity, e.g. due to immune complex 18 formation. Such toxicity has little bearing on the safety of the product in the intended 19 human host. 20 21 Although some safety testing will be required for most products, the range of tests that 22 need to be carried out should be decided on a case-by-case basis (e.g. Appendix 4) in 23 consultation with the NRA/NCL. A wide range of pharmacological, biochemical, 24 immunological, toxicological and histopathological investigative techniques should be 25 used, where appropriate, in the assessment of a product’s effect, over an appropriate 26 range of doses and, in accordance with the desired clinical indication(s), during both 27 acute and chronic exposure. However, the points made above concerning species- 28 specificity and antibody formation should always be taken into consideration. 29 30 Additional information on specific safety issues, as for example carcinogenic potential, 31 reproductive toxicity, or safety pharmacology, is provided in respective ICH safety
  35. 35. WHO/BS/2013.2213 Page 35 1 guidelines (30-32). Recommendations concerning timing and interplay of nonclinical and 2 clinical studies in drug development are given in the ICH Guidance on nonclinical safety 3 studies for the conduct for human clinical trials and marketing authorization for 4 pharmaceuticals (33) and in ICH guideline Preclinical safety evaluation of 5 biotechnology-derived pharmaceuticals (34). 6 7 B.1.1 Objectives of the nonclinical evaluation 8 The objectives of the nonclinical studies are to define pharmacological and toxicological 9 effects throughout clinical development, not only prior to initiation of human studies. 10 The primary goals are to: 1) identify an initial safe dose and subsequent dose escalation 11 schemes in humans; 2) identify potential target organs for toxicity and for the study of 12 whether such toxicity is reversible; and 3) identify safety parameters for clinical 13 monitoring. 14 15 Nonclinical evaluation should consider: 1) selection of the pharmacologically or 16 toxicologically relevant animal species; 2) age of animals; 3) physiological state of 17 animals (e.g. whether healthy/diseased animals are used, whether treatment naïve animals 18 are used); 4) weight of animals; 5) the manner of delivery, including dose, route of 19 administration, and treatment regimen; and 6) stability of the test material under the 20 conditions of use. 21 Both in vitro and in vivo studies can contribute to this characterization. 22 rDNA-derived biotherapeutics that belong structurally and pharmacologically to a (the 23 same) product class for which there is wide experience in clinical practice may need less 24 extensive toxicity testing. 25 26 B.1.2 Product development and characterization 27 In general, the product that is used in the definitive pharmacology and toxicology studies 28 should be the same as the product proposed for the initial clinical studies. However, it is 29 appreciated that during the course of development programs, changes normally occur in 30 the manufacturing process in order to improve product quality and yields. The potential
  36. 36. WHO/BS/2013.2213 Page 36 1 impact of such changes for extrapolation of the animal findings to humans should be 2 considered, including the impact of post-translational modifications. 3 The comparability of the test material should be demonstrated when a new or modified 4 manufacturing process or other significant changes in the product or formulation are 5 made in an ongoing development program. Comparability can be evaluated on the basis 6 of biochemical and biological characterization (i.e. identity, purity, stability, and potency). 7 In some cases, additional studies may be needed (i.e. PK, PD and/or safety). The 8 scientific rationale for the approach taken should be provided. 9 10 B.1.3 Good laboratory practice 11 Pivotal (toxicity) studies should be performed in compliance with good laboratory 12 practice (GLP). However, it is recognized that some studies employing specialized test 13 systems which are often needed for rDNA-derived biotherapeutics may not comply fully 14 with GLP. Areas of non-compliance should be identified and their significance evaluated 15 relative to the overall nonclinical assessment. In some cases, lack of full GLP compliance 16 does not necessarily mean that the data from these studies cannot be used to support 17 clinical trials and marketing authorization. However, justification which is supported with 18 data, such as method validation should be provided for the data quality assurance. 19 20 B.2 Pharmacodynamics 21 B.2.1 Primary and secondary pharmacodynamics/Biological activity 22 Biological activity may be evaluated using in vitro assays to determine which effects of 23 the product may be related to clinical activity. The use of cell lines and/or primary cell 24 cultures can be useful to examine the direct effects on cellular phenotype and 25 proliferation. Due to the species specificity of many rDNA-derived biotherapeutics, it is 26 important to select relevant animal species for testing (see Appendix 5). Non-human 27 primates (NHPs) are often the only pharmacologically or toxicologically relevant species; 28 however, other species should also be evaluated for relevant biological activity. In vitro 29 cell lines derived from mammalian cells can be used to predict specific aspects of in vivo 30 activity and to assess quantitatively the relative sensitivity of various species to the 31 biotherapeutics, including human. Such studies may be designed to determine, for
  37. 37. WHO/BS/2013.2213 Page 37 1 example, receptor occupancy, receptor affinity, and/or pharmacological effects, and to 2 assist in the selection of an appropriate animal species for further in vivo pharmacology 3 and toxicology studies. The combined results from in vitro and in vivo studies assist in 4 the extrapolation of the findings to humans. In vivo studies to assess pharmacological 5 activity, including defining mechanism(s) of action, are often used to support the 6 rationale for the proposed use of the product in clinical studies. When feasible, PD 7 endpoints can be incorporated into general toxicity studies (e.g. hemoglobin blood 8 concentration in repeated dose toxicity studies with erythropoetins). 9 10 B.2.2 Safety pharmacology 11 Based on the target or mechanism of action of the product, it is important to investigate 12 the potential for undesirable pharmacological activity in appropriate animal models. The 13 aim of the safety pharmacology studies is to reveal any functional effects on the major 14 physiological systems (e.g. cardiovascular, respiratory, and central nervous systems). 15 These functional indices may be investigated in separate studies or incorporated in the 16 design of toxicity studies and/or clinical studies. Investigations may include the use of 17 isolated organs or other test systems not involving intact animals. All of these studies 18 may allow for a mechanistically-based explanation of specific organ effects/toxicities, 19 which should be considered carefully with respect to their applicability for human use 20 and indication(s). 21 22 B.3 Pharmacokinetics/Toxicokinetics 23 B.3.1 General principles 24 It is difficult to establish uniform guidelines for PK studies for rDNA-derived 25 biotherapeutics. Single and multiple dose PK, toxicokinetics (TK), and tissue distribution 26 studies in relevant species are useful; however, routine studies that attempt to assess mass 27 balance are not useful. Differences in PK among animal species may have a significant 28 impact on the predictiveness of animal studies or on the assessment of dose response 29 relationships in toxicity studies. Scientific justification should be provided for the 30 selection of the animal species used for PK/TK evaluation, taking into account that the 31 PK profile in the chosen animal species should, ideally, reflect the PK profile in humans.
  38. 38. WHO/BS/2013.2213 Page 38 1 Alterations in the PK profile due to immune-mediated clearance mechanisms may affect 2 the kinetic profiles and the interpretation of the toxicity data (see also B.4.8.1). For some 3 products there may also be inherent significant delays in the expression of PD effects 4 relative to the PK profile (e.g. cytokines) or there may be prolonged expression of PD 5 effects relative to plasma levels. 6 7 PK studies should, whenever possible, utilize preparations that are representative of that 8 intended for toxicity testing and clinical use, and employ a route of administration that is 9 relevant to the anticipated clinical studies. Patterns of absorption may be influenced by 10 formulation, active substance concentration, application site, and/or application volume. 11 Whenever possible, systemic exposure should be monitored during the toxicity studies. 12 When feasible, PK/TK evaluations can be incorporated into general toxicity studies. 13 Some information on absorption, disposition and clearance in relevant animal models 14 should be available prior to clinical studies in order to predict margins of safety based 15 upon exposure and dose. Understanding the behaviour of the biotherapeutic in the 16 biologic matrix, (e.g. plasma, serum, cerebral spinal fluid) and the possible influence of 17 binding proteins is important for understanding the PD effect. 18 19 B.3.2 Assays 20 The use of one or more assay methods should be addressed on a case-by-case basis and 21 the scientific rationale should be provided. One validated method is usually considered 22 sufficient. For example, quantitation of trichloracetic acid (TCA)-precipitable 23 radioactivity following administration of a radiolabeled protein may provide adequate 24 information, but a specific assay for the analyte is preferred. Ideally, the assay methods 25 should be the same for animal and human studies. The possible influence of plasma 26 binding proteins and/or antibodies in plasma/serum on the assay performance should be 27 determined. 28 29 B.3.3 Distribution 30 Unlike small chemical drugs that readily diffuse, rDNA-derived biotherapeutics, due to 31 their molecular weight, usually do not readily diffuse, but are, following intravenous
  39. 39. WHO/BS/2013.2213 Page 39 1 application, initially confined to the vascular system. However, with time they may 2 distribute to the extravascular space by various factors, including bulk flow and active 3 transport. 4 5 As a supplement to standard tissue distribution studies, complimentary information about 6 tissue distribution of molecular targets for rDNA-derived biotherapeutics may be 7 obtained from tissue cross-reactivity (TCR) studies (see B.4.8.3). 8 9 Tissue concentrations of radioactivity and/or autoradiography data using radiolabeled 10 proteins may be difficult to interpret due to rapid in vivo protein metabolism or unstable 11 radiolabeled linkage. Care should be taken in interpreting studies using radioactive 12 tracers incorporated into specific amino acids because of recycling of amino acids into 13 non-drug related proteins/peptides. 14 15 B.3.4 Metabolism 16 The expected consequence of metabolism of rDNA-derived biotherapeutics is the 17 degradation to small peptides and individual amino acids. Therefore, the metabolic 18 pathways are generally understood. Classical biotransformation studies, as performed for 19 pharmaceuticals, are not needed. 20 21 B.4 Toxicity studies 22 B.4.1 General principles 23 Number/Gender of animals 24 The number of animals used per dose has a direct bearing on the ability to detect toxicity. 25 A small sample size may lead to failure to observe toxic events due to observed 26 frequency alone regardless of severity. The limitations that are imposed by sample size, 27 as often is the case for NHP studies, may be in part compensated by increasing the 28 frequency and duration of monitoring. Both genders should generally be used or 29 justification given for specific omissions. As an example, the minimum sample size for a 30 pivotal GLP toxicity study in NHPs is considered to be three animals per sex, and, if a
  40. 40. WHO/BS/2013.2213 Page 40 1 recovery group is included in the study, an additional minimum of two animals per sex 2 would be included. 3 4 It is desirable to apply the “3R principles” (i.e. reduction, replacement, refinement) to 5 minimize the use of animals for ethical reasons and consideration should be given to the 6 use of appropriate in vitro alternative methods for safety evaluation to reduce the use of 7 animals (35). 8 9 Administration/Dose selection and application of PK/PD principles 10 The route and frequency of administration should be as close as possible to that proposed 11 for clinical use. Consideration should be given to pharmacokinetics and bioavailability of 12 the product in the species being used, and the volume which can be safely and humanely 13 administered to the test animals. For example, the frequency of administration in 14 laboratory animals may be increased compared to the proposed schedule for the human 15 clinical studies in order to compensate for faster clearance rates or low solubility of the 16 active ingredient. In these cases, the level of exposure of the test animal relative to the 17 clinical exposure should be defined. Consideration should also be given to the effects of 18 application volume, active substance concentration, formulation, and site of 19 administration. The use of routes of administration other than those used clinically may 20 be acceptable if the route must be modified due to limited bioavailability, limitations due 21 to the route of administration, or to size/physiology of the used animal species. 22 If feasible, dosage levels should be selected to provide information on a dose-response 23 relationship, including a toxic dose and a no observed adverse effect level (NOAEL). 24 However, for oncology drugs where significant toxicity is anticipated, studies are often 25 designed to identify a “maximum tolerated dose (MTD)” rather than a NOAEL. 26 The toxicity of most rDNA-derived biotherapeutics is related to their targeted mechanism 27 of action; therefore, relatively high doses can elicit adverse effects which are apparent as 28 exaggerated pharmacology. 29 For some classes of products which show little to no toxicity it may not be possible to 30 define a specific maximum dose. In these cases, a scientific justification of the rationale 31 for the dose selection and projected multiples of human exposure should be provided. To
  41. 41. WHO/BS/2013.2213 Page 41 1 justify high dose selection, consideration should be given to the expected 2 pharmacological/physiological effects, and the intended clinical use. Where a product has 3 a lower affinity for, or potency in, the cells of the selected species than in human cells, 4 testing of higher doses may be important. The multiples of the human dose that are 5 needed to determine adequate safety margins may vary with each class of rDNA-derived 6 biotherapeutics and its clinical indication(s). 7 8 A rationale should be provided for dose selection taking into account the characteristics 9 of the dose-response relationship. PK-PD approaches (e.g. simple exposure-response 10 relationships or more complex modeling and simulation approaches) can assist in high 11 dose selection by identifying (i) a dose which provides the maximum intended 12 pharmacological effect in the selected animal species; and (ii) a dose which provides an 13 approximately 10-fold exposure multiple over the maximum exposure to be achieved in 14 the clinic. The higher of these two doses should be chosen for the high dose group in 15 nonclinical toxicity studies unless there is a justification for using a lower dose (e.g. 16 maximum feasible dose). 17 Where in vivo/ex vivo PD endpoints are not available, the high dose selection can be 18 based on PK data and available in vitro binding and/or pharmacology data. Corrections 19 for differences in target binding and in vitro pharmacological activity between the 20 nonclinical species and humans should be taken into account to adjust the exposure 21 margin over the highest anticipated clinical exposure. For example, a large relative 22 difference in binding affinity and/or in vitro potency might suggest that testing higher 23 doses in the nonclinical studies is appropriate. In the event that toxicity cannot be 24 demonstrated at the doses selected using this approach, then additional toxicity studies at 25 higher multiples of human dosing are unlikely to provide additional useful information. 26 27 Use of one or two species 28 Concerning the use of one or two species for toxicity studies, see Appendix 5. 29 30 Study duration
  42. 42. WHO/BS/2013.2213 Page 42 1 For chronic use products, repeat dose toxicity studies of 6 months duration in rodents or 2 non-rodents are usually considered sufficient, providing the high dose is selected in 3 accordance with the principles above. Studies of longer duration have not generally 4 provided useful information that changed the clinical course of development (see also 5 B.4.3). 6 For chronic use of rDNA-derived biotherapeutics developed for patients with advanced 7 cancer, see Appendix 4. 8 9 Evaluation of immunogenicity 10 Many rDNA-derived biotherapeutics intended for human use are immunogenic in 11 animals. Therefore, an immunogenicity assessment should be performed when 12 conducting repeated dose toxicity studies in order to aid in the interpretation of these 13 studies (for details, see B.4.8.1). 14 15 B.4.2 Single dose toxicity studies 16 In general, single dose toxicity studies should only be pursued in cases where significant 17 toxicity is anticipated and the information is needed to select doses for repeated dose 18 studies (33, 34). 19 Single dose studies may generate useful data to describe the relationship of dose to 20 systemic and/or local toxicity. These data can be used to select doses for repeated dose 21 toxicity studies. Information on dose-response relationships may be gathered through the 22 conduct of a single dose toxicity study, as a component of pharmacology or animal model 23 efficacy studies. The incorporation of safety pharmacology parameters in the design of 24 these studies should be considered. 25 26 B.4.3 Repeated dose toxicity studies 27 For consideration of the selection of animal species for repeated dose studies, see B.4.1. 28 The route and dosing regimen (e.g. daily versus intermittent dosing) should reflect the 29 intended clinical use or exposure. When feasible, these studies should include TK 30 measurements, but interpretation should consider the formation of possible antidrug 31 antibodies (see B.4.8.1).

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